In early May 2023, more than three years after the original declaration of COVID-19 epidemics, the World Health Organization (WHO) announced the end of the global health emergency (https://covid19.who.int/). From now on, countries should manage the virus that killed more than 6.9 million people worldwide according to routine guidelines used to monitor and treat infectious diseases.
A decreasing public interest will inevitably follow this great news. Nonetheless, the scientific and medical community is still eager to understand some unexplained features of the epidemics. Curiously, despite over 750 million cases confirmed by the WHO worldwide, roughly one-third were asymptomatic.1 Other infected individuals experienced a variety of mild to severe complications affecting the liver, kidney, and cardiovascular system. Cardiac complications included myocarditis, heart failure, thrombosis, and thrombocytopenia. Moreover, more than 11% of those hospitalized in intensive care units had myocardial ischemia or infarction. Of note, people with pre-existing cardiovascular diseases were more likely to die of COVID-19. This association has led to speculations and controversies about the paths and mechanisms followed by the virus to reach, infect, and damage cardiovascular cells.
The coronavirus virion is made up of nucleocapsid (N), membrane (M), envelope (E), and spike (S) proteins, which are structural proteins. The primary route for SARS-CoV-2 infection starts with the engagement of the S-protein with ACE-2 receptors on lung epithelial cells. Increased vascular permeability allows the virus to pass into the systemic circulation, where it can be detected in plasma and transported by monocytes and monocyte-derived macrophages (Figure 1 – left panel). Interestingly, SARS-CoV-2 viremia was associated with COVID-19 mortality.2 Moreover, the virus survives inside circulating monocytes being able to spread to different body districts.3 In addition, the S-protein was found to be present in COVID-19-infected patients’ blood early after infection and before seroconversion.4 Persistently elevated levels of the S-protein were proposed to participate in neuro-inflammation and contribute to long-COVID syndrome.5 Intriguingly, free spike antigen, probably escaped from the site of vaccine injection, was also detected in the blood of adolescents and young adults who developed post-mRNA vaccine myocarditis, a rare complication seen in 1 to 2 cases per 100,000 individuals.6 The same study showed no activation of autoantibodies. Altogether, these findings may suggest a direct pathogenic action of the S-protein.
Molecular mimicry is the sequence or structural resemblance of molecules of the host and the microbe. The phenomenon of molecular mimicry can trigger autoimmune reactions. A previous search of existing databases identified that more than two dozen hepta- and octamers in the S-protein are homologous to human proteins.7 The homology between the S-protein and self-antigens may trigger autoimmune responses and dysregulated cytokine expression leading to cardiovascular complications. Alternatively, we proposed that the S-protein may act as a ligand to induce non-infective cellular stress. We initially tested this hypothesis in cultured cardiac pericytes (PCs), exposing them to the SARS-CoV-2 wild-type strain, the α and δ variants, or the recombinant S protein alone. While being refractory to infection, PCs responded to the S-protein challenge by becoming more migratory, incapable of supporting endothelial cell network formation on Matrigel, and secreting pro-inflammatory and pro-apoptotic molecules responsible for endothelial cell death (Figure 1 – right panel).4 Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralization of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein.
As a follow-up of these in vitro studies, in the research letter “Murine studies and expressional analyses of human cardiac pericytes reveal novel trajectories of SARS-CoV-2 Spike protein-induced microvascular damage” published in Signal Transduction and Targeted Therapy, we and our coauthors present new data on the acute effects of intravenously injected S-protein on the heart microvasculature of otherwise healthy mice. We also analyzed the expressional changes caused by the S-protein in primary cultures of human cardiac PCs using bulk RNA-Sequencing. The RNA-Sequencing data were then cross-referenced with single nuclei (sn)-RNA-Sequencing datasets of COVID-19 patients’ hearts8 to determine how expressional changes after SARS-CoV-2 infection overlap with those caused by the S-protein alone. Finally, to shed light on possible therapeutic targets to antagonize the S-protein, a drug target enrichment analysis was performed using the LINCS L1000CDS and DrugBank databases.
Findings summarized in Figure 2 – left panel provide novel evidence about the SARS-CoV-2 S-protein’s direct pathogenic action on cardiac PCs and the heart’s microvasculature. Immunohistochemistry of the hearts demonstrated that the S-protein, although not altering the capillary density, increased the fraction that expresses ICAM-1, an adhesion molecule implicated in leucocyte-endothelial interactions, and remarkably reduced the PC density, coverage, and viability. Moreover, the in vivo administration of S-protein increased complement-activated C5a protein levels in peripheral blood and the heart and increased the heart’s abundance of CD45+ immune cells, specifically Ly6G/6C+ neutrophils/monocytes and F4/80+ macrophages. We hypothesize that the harmful effects observed in healthy mice three days after a single systemic injection of the S-protein might be intensified in the presence of cardiovascular risk factors and prolonged exposure. These possibilities merit further investigation.
Moreover, the S-protein modifies the transcriptional program of human cells to the virus’ advantage (Figure 2 – right panel). This new information could have significant implications for the treatment of COVID-19, for instance, using anti-S-protein antibodies to protect vascular cells. Apart from this classical approach, drug target enrichment analysis identified drugs that could protect vascular cells from the S-protein. Among the top fifty compounds, there was a prevalence of anti-tumoral, pro-apoptotic, anti-viral, anti-inflammatory, and anti-thrombotic drugs, some of which have already been trialled in COVID-19 patients. Although more research is needed to determine if pharmacological interference with the signaling emanating from the S-protein can alleviate COVID-19 outcomes, these data suggest a competitive effect of anti-inflammatory and anti-tumoral drugs. In addition, several compounds like Quercetin or ubiquitin-conjugating enzyme inhibitors could moderate inflammation by eliminating S-Protein-induced senescent cells. Such remedies might also help treat the rare cases that manifest post-vaccinal myocarditis.
1 Sah, P. et al. Asymptomatic SARS-CoV-2 infection: A systematic review and meta-analysis. Proceedings of the National Academy of Sciences of the United States of America 118 (2021).
2 Giacomelli, A. et al. SARS-CoV-2 viremia and COVID-19 mortality: A prospective observational study. PLoS One 18, e0281052 (2023).
3 Percivalle, E. et al. Macrophages and Monocytes: "Trojan Horses" in COVID-19. Viruses 13 (2021).
4 Avolio, E. et al. The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease. Clin Sci (Lond) 135, 2667-2689 (2021).
5 Theoharides, T. C. Could SARS-CoV-2 Spike Protein Be Responsible for Long-COVID Syndrome? Mol Neurobiol 59, 1850-1861 (2022).
6 Yonker, L. M. et al. Circulating Spike Protein Detected in Post-COVID-19 mRNA Vaccine Myocarditis. Circulation 147, 867-876 (2023).
7 Khavinson, V., Terekhov, A., Kormilets, D. & Maryanovich, A. Homology between SARS CoV-2 and human proteins. Sci Rep 11, 17199 (2021).
8 Delorey, T. M. et al. COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets. Nature 595, 107-113 (2021).